GMSCs encapsulated in soft 3D-scaffolds enable nerve regeneration via immunomodulation and neurite outgrowth stimulation

Faizan I. Motiwala

Motiwala, Faizan I., Shi, Shihong, Shanti, Rabie M.
Faculty / Advisor: Zhang, Qunzhou, Le, Anh D.
University of Pennsylvania School of Dental Medicine, Department of Oral & Maxillofacial Surgery/Pharmacology


Gingiva-derived mesenchymal stem cells (GMSCs) represent an easily accessible source of post-natal stem cells of a neural crest-origin. They possess multipotent and potent immunomodulatory/anti-inflammatory capabilities. Recently, we demonstrated that GMSCs could be non-genetically induced into neural crest stem-like cells (NCSCs) under defined culture conditions. In this study, we investigated whether 3D-scaffolds could promote the enrichment of NCSC-like cell properties in GMSCs via a mechano-sensing stiffness mechanism.


GMSCs (2×106/mL) were encapsulated in methacrylated collagen hydrogel (4mg/mL) and allowed to crosslink at 37°C for 30 minutes and then cultured under regular MSC culture conditions. Immunofluorescence (IF) study, Western blot, and qRT-PCR were performed to compare the expression of NCSC-related genes between 3D- and 2D-cultured GMSCs. GMSCs in hydrogels were cocultured with M0 macrophages followed by stimulation with lipopolysaccharide (LPS). The secretion of pro-inflammatory cytokines, IL-1β and TNF-α, was determined by ELISA. PC12 were seeded on D-laminin plates and cultured in the presence or absence of NGF or GMSCs encapsulated in collagen hydrogels for 48h and then neurite outgrowth was assessed.


GMSCs encapsulated in collagen hydrogels showed significant increased expression of a panel of NSCS-related genes, such as p75NTR, SOX9, ERRFI1, GDNF, and NTF3, in comparison to those in 2D-culture. Meanwhile, GMSCs encapsulated in collagen hydrogels exhibited potent inhibitory effect on the secretion of pro-inflammatory cytokines by macrophages. GMSCs encapsulated in hydrogels also remarkably induced neurite outgrowth in PC12 cells.


These results have demonstrated that GMSCs were enriched with NSCS-like properties and maintained their immunosuppressive effects on M1 macrophages when cultured in soft 3D collagen hydrogels, suggesting that soft substrate stiffness can direct reprogramming of GMSCs into NCSC-like cells. Taken together, our findings support that NCSCs non-genetically reprogrammed from GMSCs would be a promising and easily accessible source of supportive cells with translational potentials in tissue engineering/regenerative medicine.